We are interested in determing the mechanisms by which human cells control spontaneous and induced mutation rates. We have previously shown tht the major replicative DNA polymerase in human cells, when purified as a relatively simple DNA polymerase of limited complexity, is not accurate enough during DNA synthesis reactions in vitro to account for low spontaneous mutations rates in vivo. However, it is well established the replication involves the concerted action of a number of proteins. We are therefore examining the fidelity of semiconservative bidirectional DNA replication by a human HeLa cell replication complex. Our initial results, obtained in a forward mutation assay capable of detecting a variety of errors, indicating that this replication system was at least 20-fold more accurate than the purified replicative DNA polymerase alone. This observation has been substantiated by several control experiments, necessitated by the complicated nature of the system. In order to more accurately assess the fidelity of replication, mutagenesis vectors have been constructed and used to monitor the base substitution and frameshift fidelity of the replication complex in highly sensitive reversion assays. Initial results with these assays demonstrate that for minus-one frameshift errors at reiterated template sequences, DNA synthesis by the replication complex is at least 500-fold more accurate than purified DNA polymerase alpha. However, the base substitution fidelity of the replication complex, while high, is still insufficient to explain the extremely low mutation rates observed in intact cells. This suggests that additional mechanisms for maintaining accurate DNA synthesis are present within the cell. We are currently dissecting the replication system into its component parts and testing the effects of individual subunits on specific types of mutation events using the highly sensitive reversion assays. We intend to focus on mechanisms that contribute to fidelity, including base selectivity, exonucleolytic proofreading and post-replication error correction.